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Thylakoid DeltapH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport.

Cline K, Mori H - J. Cell Biol. (2001)

Bottom Line: Thylakoid-bound precursor proteins were also associated with an approximately 700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106.Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane.These results indicate that precursor binding to the cpTatC-Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.

View Article: PubMed Central - PubMed

Affiliation: Horticultural Sciences and Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611, USA. kcline@ufl.edu

ABSTRACT
The thylakoid DeltapH-dependent pathway transports folded proteins with twin arginine-containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC-Hcf106 complex serves as receptor for specific binding of twin arginine-containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an approximately 700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an approximately 700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC-Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of DeltapH. These results indicate that precursor binding to the cpTatC-Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.

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Chemical cross-linking of precursor-bound thylakoids. (A) In vitro translated DT17 was incubated with thylakoid membranes in a binding assay. The membranes were washed and treated with varying concentrations of DSP or DTSSP as described in Materials and methods. Concentrations of cross-linker (mM) are depicted at top. The membranes were then analyzed by SDS-PAGE and fluorography. (B) A 0.1-mM DSP-treated sample, equivalent to 0.33 mg total chlorophyll, was denatured with SDS and divided into six aliquots, five of which were subjected to immunoprecipitation under denaturing conditions with IgGs cross-linked to beads as designated above the panel. Immunoprecipitated samples were released with SDS and analyzed by SDS-PAGE and fluorography as described in Materials and methods. (C) A portion of each sample in B was treated with 2.5% mercaptoethanol to cleave the cross-linking agent and was then subjected to SDS-PAGE and fluorography. Each sample in B is equivalent to 6 μg chlorophyll, and each sample in C is equivalent to 4 μg chlorophyll of starting thylakoids.
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fig6: Chemical cross-linking of precursor-bound thylakoids. (A) In vitro translated DT17 was incubated with thylakoid membranes in a binding assay. The membranes were washed and treated with varying concentrations of DSP or DTSSP as described in Materials and methods. Concentrations of cross-linker (mM) are depicted at top. The membranes were then analyzed by SDS-PAGE and fluorography. (B) A 0.1-mM DSP-treated sample, equivalent to 0.33 mg total chlorophyll, was denatured with SDS and divided into six aliquots, five of which were subjected to immunoprecipitation under denaturing conditions with IgGs cross-linked to beads as designated above the panel. Immunoprecipitated samples were released with SDS and analyzed by SDS-PAGE and fluorography as described in Materials and methods. (C) A portion of each sample in B was treated with 2.5% mercaptoethanol to cleave the cross-linking agent and was then subjected to SDS-PAGE and fluorography. Each sample in B is equivalent to 6 μg chlorophyll, and each sample in C is equivalent to 4 μg chlorophyll of starting thylakoids.

Mentions: The primary objective for cross-linking of precursor-bound thylakoids was to identify components that directly interact with the precursor. The precursor DT17 (see below) was used as it yielded a high level of cross-linking products in preliminary experiments with DSP and DTSSP. A concentration series of each cross-linker identified amounts that produced distinctive bands representing putative 1:1 adducts between precursors and nearest neighbors (Fig. 6 A, lanes 2–4 and 6–8). Notable products were those at ∼39, ∼48, and ∼75 kD. The relative amounts of these products varied with the cross-linker, but the band at ∼48 kD was uniformly present. Because Hcf106 migrates on SDS gels at ∼32 kD, cpTatC at ∼33 kD, and DT17 at ∼20 kD, the 48-kD band could conceivably represent a cpTatC-DT17 or Hcf106-DT17 cross-linking product. In addition to distinct bands, very large cross-linking products were also apparent (Fig. 6 A, lanes 2–4).


Thylakoid DeltapH-dependent precursor proteins bind to a cpTatC-Hcf106 complex before Tha4-dependent transport.

Cline K, Mori H - J. Cell Biol. (2001)

Chemical cross-linking of precursor-bound thylakoids. (A) In vitro translated DT17 was incubated with thylakoid membranes in a binding assay. The membranes were washed and treated with varying concentrations of DSP or DTSSP as described in Materials and methods. Concentrations of cross-linker (mM) are depicted at top. The membranes were then analyzed by SDS-PAGE and fluorography. (B) A 0.1-mM DSP-treated sample, equivalent to 0.33 mg total chlorophyll, was denatured with SDS and divided into six aliquots, five of which were subjected to immunoprecipitation under denaturing conditions with IgGs cross-linked to beads as designated above the panel. Immunoprecipitated samples were released with SDS and analyzed by SDS-PAGE and fluorography as described in Materials and methods. (C) A portion of each sample in B was treated with 2.5% mercaptoethanol to cleave the cross-linking agent and was then subjected to SDS-PAGE and fluorography. Each sample in B is equivalent to 6 μg chlorophyll, and each sample in C is equivalent to 4 μg chlorophyll of starting thylakoids.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2196467&req=5

fig6: Chemical cross-linking of precursor-bound thylakoids. (A) In vitro translated DT17 was incubated with thylakoid membranes in a binding assay. The membranes were washed and treated with varying concentrations of DSP or DTSSP as described in Materials and methods. Concentrations of cross-linker (mM) are depicted at top. The membranes were then analyzed by SDS-PAGE and fluorography. (B) A 0.1-mM DSP-treated sample, equivalent to 0.33 mg total chlorophyll, was denatured with SDS and divided into six aliquots, five of which were subjected to immunoprecipitation under denaturing conditions with IgGs cross-linked to beads as designated above the panel. Immunoprecipitated samples were released with SDS and analyzed by SDS-PAGE and fluorography as described in Materials and methods. (C) A portion of each sample in B was treated with 2.5% mercaptoethanol to cleave the cross-linking agent and was then subjected to SDS-PAGE and fluorography. Each sample in B is equivalent to 6 μg chlorophyll, and each sample in C is equivalent to 4 μg chlorophyll of starting thylakoids.
Mentions: The primary objective for cross-linking of precursor-bound thylakoids was to identify components that directly interact with the precursor. The precursor DT17 (see below) was used as it yielded a high level of cross-linking products in preliminary experiments with DSP and DTSSP. A concentration series of each cross-linker identified amounts that produced distinctive bands representing putative 1:1 adducts between precursors and nearest neighbors (Fig. 6 A, lanes 2–4 and 6–8). Notable products were those at ∼39, ∼48, and ∼75 kD. The relative amounts of these products varied with the cross-linker, but the band at ∼48 kD was uniformly present. Because Hcf106 migrates on SDS gels at ∼32 kD, cpTatC at ∼33 kD, and DT17 at ∼20 kD, the 48-kD band could conceivably represent a cpTatC-DT17 or Hcf106-DT17 cross-linking product. In addition to distinct bands, very large cross-linking products were also apparent (Fig. 6 A, lanes 2–4).

Bottom Line: Thylakoid-bound precursor proteins were also associated with an approximately 700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106.Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane.These results indicate that precursor binding to the cpTatC-Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.

View Article: PubMed Central - PubMed

Affiliation: Horticultural Sciences and Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611, USA. kcline@ufl.edu

ABSTRACT
The thylakoid DeltapH-dependent pathway transports folded proteins with twin arginine-containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC-Hcf106 complex serves as receptor for specific binding of twin arginine-containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an approximately 700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an approximately 700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC-Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of DeltapH. These results indicate that precursor binding to the cpTatC-Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.

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